1. Technical Field
This invention relates in general to the quantitive determination of minute amounts of substantially opaque insoluble colored light absorptive substances, and more particularly to a highly sensitive light transmittance type optical system useful in conjunction with analytical assays productive of such substances, for the rapid determination of the presence of minute amounts of such substances at levels which have heretofore been undetectable by the use of conventional techniques.
2. Background Art
Assays such as immunological assays and nucleic acid hybridization probe assays, have been the subject of much research and development activity in recent years. These and other assays have employed labelled entities which result in the production of a light absorptive colored end product as an indication of the presence of a particular analyte in a fluid specimen assayed.
In such assays it is common to bind to a portion of the analyte a reporter component having an enzymatic label capable of catalyzing the production of a colored light absorptive end product when contacted by a suitable substrate, and the quantity of the analyte in the specimen is usually determined by subjecting the colored end product to reflectance photometry or spectrophotometric light absorbence measurement.
Various formats have been developed for assay performance, and much effort has been expended to maximize the efficiency thereof in order to be able to quantitate lower and lower levels of analyte in a specimen. However, various probems have been encountered in such development.
In those assays in which the presence of a particular analyte in a specimen is indicated by the presence of a quantity of a soluble colored pigment in a clear solution, it is standard practice to measure the absorbence of the pigment spectrophotometrically as mentioned above. In this procedure, monochomatric light is directed through the solution of interest and toward a light detector such as a photodiode. The amount of the analyte is determined as a function of the amount of light absorbed by the colored pigment, as measured by the difference between the amount of incident monochromatic light and the amount of light reaching the light detector. More particularly, the amount of light absorbed is described by the Beer-Lambert Law which is often expressed as ##EQU1## in which I.sub.o is the incident monochromatic light
I is the transmitted light PA1 e is the molar extinction coefficient PA1 l is the length of the light path through the absorber, and PA1 C is the concentration of the absorber.
It will be observed that in the above equation the absorbence is proportional to the molar extinction coefficient, the length of the light path, and the concentration of the absorber. Thus, when measuring the concentration of an analyte in a liquid sample having a low molar extinction coefficient, or a sample having a very low concentration of analyte, the concentration is sometimes increased through evaporation of the sample and/or the sample is placed in a cuvette with a very long light path length. However, neither of these alternatives provides a practical solution in those instances where the sample, Without evaporation, has a volume too small to fill a standard cuvette.
As reported by Butler, W. L., in J. Opt. Soc. Am., Vol. 52, 292-299, (1962), one approach to the small sample problem involves the intensification of absorption by light scatter. In the reported procedure, various amounts of light scattering particles, e.g., particles of calcium carbonate, were added to a clear solution of soluble pigment. This introduced an additional factor into the absorbence equation above illucidated, so that A=.beta.elC, wherein .beta. represents the increased light path length due to light scatter from the scattering particles. Butler found that the value for .beta. was as much as 100, indicating that light path length through the optical cell was 100 times longer than it was before the scattering particles were introduced into the solution. He also found that the absorbence of the sample in the light scattering mixture was substantially greater than that of the same amount of sample added to plain water. The magnitude of the increase is the value of .beta..
Current developments in nucleic acid hybridization assays, as well as immunological assays, include the detection of a conjugate of a labelled component with a component bound to a nitrocellulose filter. Such detection involves the measurement of the intensity of visibly colored areas on the filter as indicative of the amount of analyte in a test sample. One such procedure is reported by Renz et al in Nucleic Acids Res., Vol. 12, 3435-3444, (1894). In the reported procedure, nucleic acid probes labelled with peroxidase or alkaline phosphatase were annealed to nitrocellulose bound target nucleic acid. The presence of the substrate caused the label to produce a pigmentation on the paper, the intensity of which was read spectrophotometrically.
Certain immunological assays also involve similar procedures. In one such assay, a liquid specimen containing one component of an antibody/antigen couple, e.g., an antigenic analyte, is applied to a nitrocellulose membrane or the like having bound thereto a capture antibody specific for the analyte. This causes binding of the antigenic analyte to the capture antibody and thereby to the membrane. A solution containing a reporter component comprising an enzymatically labelled antibody to said antigenic analyte is run through or over the membrane to cause formation of a conjugate of said labelled antibody with the antigenic analyte. Any unbound labelled antibody is then removed from the membrane, as by washing, and a solution containing a substrate for the enzymatic antibody label is then run over or through the membrane to produce a pigmentation on the membrane indicative of the presence of the analyte in the specimen assayed. The pigmentation intensity is then read by reflectance spectrophotometry.
In order to achieve greater sensitivity in assays of the general type just described, it has become increasingly important to detect the presence of smaller and smaller quantities of a colored end product to thereby correspondingly detect smaller and smaller quantities of analyte in a test sample. However, there are a variety of problems associated with such assays, some of which are nonuniform color, chemical incompatability, poor membrane wetting, poor optical quality, and ineffective binding.